The studies outlined in this proposal are aimed at understanding the role of VEGF in retinal pigment epithelium (RPE) - choriocapillaris interactions. Significant evidence supports a role for the RPE in the development and maintenance of the choroidal vasculature, and interactions between RPE cells and the choroidal vasculature are central in age-related macular degeneration (AMD). We hypothesize that tightly regulated expression of VEGF by RPE is necessary for the integrity and maintenance of the adult choroidal vasculature. To test this hypothesis, we propose the following. (1) To examine the role of RPE-derived VEGF in the stability of the adult choriocapillaris. Mice engineered for the inducible deletion of VEGF in the RPE will be generated to determine if VEGF derived from RPE is necessary to the integrity of the choriocapillaris in the adult. In addition, we have observed that transgenic mice that express only VEGF188 display an age-dependent degeneration of the choriocapillaris and RPE, which has many of the features of dry/atrophic AMD. The phenotype of these mouse models will be characterized using light and electron microscopy, fundus photography and ERG. (2) To determine if VEGF has autocrine effects on RPE in vivo and in vitro. RPE cells express VEGFR2 in adult, but not during development. This fact, coupled with the continuous expression of VEGF by RPE suggests a developmentally regulated autocrine role for VEGF. A possible role for VEGF in RPE will be investigated both in vitro using siRNA and in vivo by RPE- specific deletion of VEGFR2. (3) To elucidate the molecular regulation of physiologic VEGF expression by RPE. The mechanisms of VEGF expression in adult RPE will be investigated by co-expressing candidate transcription factors along with VEGF promoter-luciferase constructs. The role of transcription factors identified in promoter-reporter assays will be determined in RPE by knockdown using siRNA. The involvement of particular promoter binding sites will be verified by mutagenesis studies and by chromatin immunoprecipitation studies. Results of these studies will not only provide important insights into the control of VEGF expression, but should also be useful in the development of novel anti-VEGF therapies that can target VEGF involved in pathologic angiogenesis while sparing physiological VEGF expression. PUBLIC HEALTH RELEVANCE: Interactions between the retinal pigment epithelium and choroidal circulation are vital to normal retinal function and to pathologies such as age-related macular degeneration (AMD). Vascular endothelial growth factor (VEGF) is likely central to both. Understanding the role of VEGF in the maintenance of the choriocapillaris and the pigment epithelium is important to understanding the pathogenesis of AMD. In light of VEGF's role in vascular maintenance, current therapies aimed at VEGF neutralization in the eye for wet AMD may have unwanted side effects. Knowledge of the mechanisms of VEGF regulation in RPE under normal conditions may provide an opportunity to develop novel anti-VEGF therapies to inhibit the VEGF that mediates pathologic choroidal neovascularization, while sparing physiological VEGF expression.